In this proposal, we will use two biological models to investigate DNA methylation, which plays key roles in oncogenesis yet is absent in many model systems. One system we utilize is the ciliate Oxytricha trifallax, which was only recently discovered to use cytosine methylation in a remarkable genome rearrangement process. Aim 1 is a mechanistic investigation of a novel methyltransferase complex biochemically purified from Oxytricha nuclear lysate. The role of one complex member, a DNA-PKcs homolog, in detecting aberrant chromosomes and guiding DNA methylation is a particular focus. Aim 2 is a combined mouse cell line (10T1/2) and Oxytricha screen of FDA-approved clinical compounds to identify those with methylation-modulating properties. Both Oxytricha and mouse 10T1/2 cells provide robust DNA methylation-driven phenotypes, allowing rapid screening for novel active compounds. Given the lack of DNA methylation in worms, flies, and yeast, these model systems provide ideal platforms for rapid scientific advance and for drug screening and repurposing efforts. Immediate and Long-term Career Goals My ultimate career goal is to be an innovative tenure-track faculty member who makes important contributions to basic and clinical research in the exciting field of epigenetics. Central to this goal is a shift from purely basic research to a combination of basic and applied research centered on the unique biology of Oxytricha and other model systems. The proposed research elaborates the advantages of Oxytricha into both clinical and basic research trajectories, and also rekindles an older mouse cell line, 10T1/2, for methylation drug screening, a novel application. This work will meet a deep need for model systems to study DNA methylation with clinical applications. In order to transition into an independent faculty position, I will perform the basic research described in Specific Aim 1 immediately, culminating in the publication of a manuscript describing the novel methylation machinery in Oxytricha. Year 2 will be dedicated to performing the chemical screen for methylation inhibitors (Aim 2) and writing an R01 grant application with the resultant data. I hope to have the screen finished by the end of Year 2, leaving Year 3 for a methylation enhancer screen and follow-up on mechanisms of action of the interesting hits, validation studies, and manuscript writing. This Career Development Award will allow the protected time necessary to establish publication and grant support necessary for a fast start in running my own laboratory.